X inactivation results in the silencing of most genes on the mammalian X chromosome. However, some genes escape silencing and are thus expressed from both alleles in females. Turner syndrome is thought to result in part from haploinsufficiency of these escape genes. We have previously shown that there are few escape genes in mouse and that silencing precedes escape in early embryogenesis. Each mouse escape gene is embedded within silenced chromatin, while human escape genes are often grouped in larger domains. Our search for chromatin boundary elements between an escape gene (Smcx) and an adjacent activated gene has identified CTCF, a zinc finger protein known to have insulator function, as binding to this region in mouse. CTCF does not bind to the corresponding region in human where the two adjacent genes escape. We have also found that, even in early embryogenesis, DNA methylation is very low at Smcx CpG island including at the CTCF binding sites, suggesting that this protein may prevent the establishment of DNA methylation. Our goal is to characterize escape domains on the X chromosome and to investigate the role of CTCF and epigenetic modifications in the establishment of domains. In the present proposal we outline experiments to (1) search for insulator elements at other regions of the genome that separate escape genes from silenced genes; (2) to examine the chromatin structure of a well defined region of escape and surrounding regions in terms of histone modifications and intergenic transcripts and to follow epigenetic modifications during embryogenesis (3) to construct mice containing mutations at the endogenous CTCF binding sites and mice with inserts of large X chromosome segments containing escape genes, with and without mutations at the associated CTCF binding sites. These functional approaches are aimed at determining the chromatin configuration of escape domains and the role of CTCF in escape and DNA methylation.